Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens

ABSTRACT Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-β-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens, while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N2-fixing efficiency of the peanut nodules is three times higher than that of S. flavescens. By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Bradyrhizobium guangxiense (2) and others (3, 4). Inside the root nodules of peanut, 47 the bradyrhizobia differentiate into spherical morphotype of bacteroids (5, 6). 48 The medicinal legume Sophora flavescens (7, 8) (common name "Kushen" in 49 Chinese that means "bitter ginseng") is an extremely promiscuous host plant  to culture the rhizobial strain at 28 °C. Water-agar (0.8%, g/w) was used to support the 94 5 germination (28 °C, two to three days) of the plant seeds after sterilization using 95 2.5-3.0% (w/v) sodium hypochlorite (NaClO) for 5 min. Seeds of S. flavescens were 96 treated using concentrated sulfuric acid for 20 min previous to the surface-sterilization 97 to remove the waxy layer of seed coat. Sterilized deionized water was used to rinse 98 the surface sterilized seeds for 6-7 times for removing the disinfectant. Sterilized

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After activation on YM agar medium, the rhizobial strain was transferred into TY 110 liquid broth and cultured at 28 °C with shaking (160 rpm) to density of 4.5-5.5 111 (OD 600nm ). The cultures were collected by centrifugation (4000 rpm, 15-20 min). The 112 collected cells were used to extract the RNA immediately, or the liquid nitrogen was 113 added into the centrifuge tube and then stored in a refrigerator at -80 °C for 114 subsequent RNA extraction.

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Collection of root nodules used for RNA extraction 116 To prevent the degradation of target RNA, the collected nodules of peanut and S. 117 flavescens should be immediately transferred into a 50 ml centrifuge tube filled with 118 liquid nitrogen, and the nodules should always be kept in the liquid nitrogen 119 environment during the collection process. After collecting the nodules, the centrifuge 120 6 tubes were transferred immediately into a refrigerator at -80 °C.

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Regents and kit used in RNA extraction, PCR and sequencing 122 The following reagents were used to extract RNA and for PCR. Total  About 0.1 g rhizobial cells were added into mortar pre-cooled by liquid nitrogen and 132 ground into powder thoroughly. Then, the powder was transferred into RNase-free 133 tube containing 1 mL RNAiso Plus reagent (Takara, #9108) and mixed thoroughly.

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After being kept for 5-10 min at room temperature, the mixture was centrifuged at 135 4 °C for 15 min (12,000 rpm). The supernatant was transferred to a clean 1.5 mL 136 centrifuge tube and was mixed with 2 ml chloroform. After another 5-10 min 137 incubation at room temperature, it was centrifuged again at the same condition. The 138 supernatant (about 400 μL) was transferred into another new 1.5 mL centrifuge tube.

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One volume of isopropanol and one volume of high salt precipitation solution were 140 added to the supernatant phase. The solution was gently reversed, mixed, and 141 incubated at room temperature for 10 minutes, and then incubated at -20 °C overnight.

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After centrifugation at 4 °C for 10 min at 12,000 rpm, the supernatant was removed 143 without touching the precipitate. One mL of 70% ethanol was added and centrifuged 144 at 4 °C for 10 min at 12,000 RMP. After discarding the supernatant, 1 mL of absolute 145 ethanol was added to each tube for storage and transportation. When the RNA was 146 7 ready to use, the tube was centrifuged, the ethanol phase was discarded, and the tube 147 was dried a minute at room temperature, then small amount of DEPE pure water 148 (about 50 μL) was added to dissolve RNA.    Then it was dehydrated subsequently with 30%, 50%, 70%, 80%, 90% and 100%   HiSeq TM 2500 platform. The reading length was 100 bp and they were sequenced by 211 paired ends. The raw data obtained after sequencing was raw reads and it should 212 remove the reads with adapters, the sequences containing more than 5% N bases, and 213 low-quality reads. The data obtained from the company were called clean reads.         Sequencing data and mapping to known genome sequence 280 The RIN values of all RNA samples were above 7.5. All RNA samples met the 281 high-quality requirements for RNA-seq database construction and they were sent to 282 the company to build the database and subsequent sequencing.

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The sequence data were used to map the reference sequence for B. arachidis CCBAU  for ureA and ureB, respectively) but they were not expressed significantly in peanut 340 bacteroids ( Fig. 4A and 4B). On the contrary, of these 30 down-regulated expression 341 genes, total 10 genes were common in both kinds of the bacteroids and most of them 342 (7) were hypothetical proteins (HP) (Fig. 4).

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(1) Expression of taxis, motivity and flagellin related genes 344 The expression of genes involved in chemotaxis, motivity and flagellin were

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(3) Expression of nitrogen fixation genes 369 The expression of nif and fix genes in symbiotic nitrogen fixation process were 370 significantly increased both in the bacteroids of peanut and S. flavescens (Fig. 5C).

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The expression of structure genes for nitrogenase (nifHDK) rank the first three tops The genes expression that participated in the fatty acid metabolism in the bacteroids 400 of both hosts was not active or down-regulated (Fig. 5F).

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Through the comparison of COG functional categories (Fig. 3), the distribution of

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The PHB accumulation was found mainly in bacteroids of S. flavescens nodules but 513 few was observed in bacteroids of peanut nodules through the observation under TEM 514 (Fig. 1A4) and the result was consistent with the up-or down-regulated gene 515 expression of PHB syntheses (Fig. 5G), respectively, in these two kinds of bacteroids.

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In addition, the PHB content was clearly not related with nitrogenase activity as 517 bacteroids in peanut nodules had few or no PHB, but the nodules had higher N 2 -fixing  The process of submission is smoothly and conveniently. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
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• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.
Done. Each figure was a separate file.

R:
Ok, I will join the membership.
Thank you for submitting your paper to Microbiology Spectrum. The nodule numbers were supplied and added to the text (line 266-267).
The Y-axis of the barplot for ARA data was revised to production of C 2 H 4 so it would be appropriate to represent the acetylene reduction for the production of C 2 H 4 was the characterization of nitrogenase activity.
More information about the nodule section were supplied including the size of the bacteroids (Fig. 1C) and the area of bacteroid section (text, line 294-295).
And the three segments have been combined into one section. Here also the text reads flat and no specific message is delivered.

R:
Thanks for your kind comments. The legend for this Fig. 3 was improved and more information were provided as following: hypogaea vs. free-living bacteria. Gene expressions were down-regulated or up-regulated respectively in the two kinds of bacteroids and were classified into different COG categories compared with the free-living bacteria. Numbers of DEGs were counted. The plot was constructed using functions ggplot, ggarrange of the "ggplot2", "ggpubr" packages in R language.
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